The removal of introns from messenger RNA precursors by splicing is a required step in the expression of most higher eukaryotic genes. The regulation of this process contributes to the processes of development and tissue-specific gene expression. The general aim of this work is to understand the details of the process by which sites of splicing are identified by cellular factors and brought together in a functional spliceosome complex. The identities of the cellular factors and the nature of their interactions with splice sites are poorly understood yet underlie the specificity, fidelity and regulation of the splicing process. We have been addressing these questions by 1) developing techniques to define the sites of interaction between cellular factors and pre-mRNA substrates in in vitro systems and 2) biochemically fractionating the in vitro systems to identify, purify and characterize the factors that interact at these sites. The specific aims of the proposed research are to: 1) Characterize the complexes in which protection of pre-mRNA substrates has been detected by the iron-EDTA RNA footprinting technique. Physical resolution of spliceosomes and sub-spliceosomal complexes and modified reaction conditions will be used to correlate protections with the association of particular cellular factors. Construction and analysis of specific mutations in protected regions will be used to investigate the sequence specificity of the interactions. 2) Use phosphorothioate substitution in pre-mRNA substrates to determine sites of RNA - factor interactions in a modification/interference assay. Phosphorothioate substituted RNA will also be used to determine the stereochemistry of the two cleavage/ligation reactions in the splicing mechanism. 3) Define the interactions between and among the pre-mRNA, snRNPs and other factors in spliceosomes and sub-spliceosomal complexes. These will be studied by indirect iron-EDTA footprinting of snRNAs alone and in splicing complexes, analysis of RNA - RNA interactions by crosslinking methods and determination of the sites of crosslinking using linker assisted amplification of reverse transcripts of crosslinked RNA. 4) Characterize previously purified splice site binding proteins and identify and purify new activities identified in the above investigations. Molecular and immunological probes will be developed from the purified proteins. The function of these proteins in the process of splice site identification will then be determined and their interactions with snRNPs and other components will be addressed.